Abstract
BackgroundStudies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls.ResultsThe number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups.ConclusionsDifferences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.
Highlights
Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene
Number of sequences and amplicon sequence variants (ASVs) We began our analyses with a comparison of the number of sequences and amplicon sequence variants (ASVs) retained at each step when processing through the bioinformatic pipeline (Fig. 1)
The greatest decrease in ASV number occurred after the removal of small ASVs, for which the number of sequences was calculated to be less than 0.005% of the total number of sequences on the same run (Fig. 1, step 3)
Summary
Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. Common to all amplicon based protocols is the application of the polymerase chain reaction (PCR) for i) amplification of the target marker gene to be sequenced and ii) the addition of index sequences necessary for sample multiplexing. These steps can be performed in a single PCR or in two separate PCRs. No study has addressed whether the increased number of laboratory processing steps associated with a 2-step PCR protocol, will leave samples more vulnerable to bacterial DNA contamination from the laboratory than when following a 1-step PCR protocol. We predicted that while samples with a high bacterial load (i.e. upper airway samples) would be able to buffer against protocol effects resulting from differences in contamination levels, samples with a low bacterial load (i.e. lower airway samples) would not be resistant to these effects
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